Cells are the basic unit of all living organisms. The one common attribute of almost all cells is that they are surrounded (or bounded) by a cytoplasmic membrane. This membrane harbors the internal contents of the cell and regulates the movement of substances into and out of the cell. Only those molecules that can diffuse across the membrane or are transported across it can move into and out of the cell. Some can pass through the lipid core of the membrane, but others must pass through pores. Still other molecules must cross the membrane attached to carriers in an energy dependent manner. Likewise, the nucleus and other cellular organelles have membranes to regulate the flow of molecules into and out of the organelle.
Fixation is a chemical process that “sets” cellular molecules in place so that the cell or tissue can then be studied. Most agents that are used as fixatives (e.g., alcohols such as ethanol and aldehydes such as paraformaldehyde) work by crosslinking cellular molecules, especially proteins. This crosslinking process prevents the degradation of the cellular structure. Various fixatives are better suited for the preservation of different cellular molecules and structures or for different detection methods. The fixative chosen for any particular purpose will be determined by the nature of that purpose.
Unfortunately, the current methods of fixation often hamper the subsequent ability of a researcher or clinician to detect internal cellular components. In other words, the very thing that prevents the degradation of the cell, fixation, can also set up a barrier to the many types of research and diagnosis that rely on larger sized detection molecules. Because of this, efforts have been made to permeabilize cells or make channels after fixation.
Current methods of permeabilizing the cell membrane after fixation are not effective for all specimens, are too rigorous (thus, destroying the structures to be studied) and/or require expensive equipment. For example, Hoffman, et al. (U.S. Pat. No. 6,835,393) disclose the use of polycarboxylic acid polymers and pH for disrupting cell membranes only for use in non-fixed samples. Connelly, et al., (U.S. Pat. Nos. 5,597,688 and 5,422,277) disclose the use of a composition with 2,4-dinitrobenzene sulfonic acid, 2,4-dinitrobenzoic acid or 2,4-dinitrophenol for both cell membrane fixation and permeabilization but these compositions limit the researcher's or clinician's choice of fixative and, thus, limits necessary assay flexibility. Mechanical methods such as sonication, electroporation, etc. usually only work on unfixed samples and require expensive equipment.
Furthermore, the available research and diagnostic methods of the prior art for many cellular targets such as pathologies depends on microscopic evaluations, cellular morphological parameters, staining characteristics and the presence or absence of certain targets. However, many of these diagnostic methods are not entirely accurate or sufficiently sensitive.
What is needed are compositions and methods for the improved permeability of cell membranes of specimens to foreign particles such as labeled detection molecules. Furthermore, what is needed are compositions and methods for the improved detection of cellular targets and pathogens.